Chemiluminescent 1,2-dioxetanes

ABSTRACT

A method of generating light through chemiluminescence involves providing a stable 1,2-dioxetane of the formula: 
                         
Wherein (a) R 1  and R 2  are each, individually, a chemical reactive site or when fused together form a chemical reactive site, and R 3  and R 4  are each, individually, a chemical reactive site or when fused together form a chemical reactive or (b) R 1  has at least two hetero atoms with chemical reactive site and R 3  and R 4  are inactive site and R 2  is a chemical reactive site.

CROSS REFERENCE TO RELATED APPLICATION

This application is a national application based on PCT Application,Ser. No. 99/20590, filed Sep. 8, 1999, which is a completion applicationof U.S. provisional patent application Ser. No. 6-0/099693, filed Sep.8, 1998 for “Chemiluminescent 1,2 dioxetanes”, the disclosures of whichare hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to chemiluminescent compounds. Moreparticularly, the present invention concerns stable, triggerablechemiluminescent 1,2-dioxetanes. Even more particularly, the presentinvention concerns new chemiluminescent 1,2-dioxetane compounds derivedfrom the oxidation of novel alkenes prepared by the coupling ofsubstituted aromatic esters or ketones and spiro-fused ketones with orwithout a π-electron system or a carbon-carbon double bond in the ring.

2. Prior Art

Chemiluminescent compounds, their preparation and their uses have beenlong documented in the prior art. These “high energy” molecules storesufficient energy to generate, or fragmentation, electronically excitedcarbonyl products which are responsible for the observedchemiluminescence. Dioxetanes and especially, 1,2-dioxitanes andeminently useful to detect the presence, as well as the absence, ofcertain enzymes in fluids such as blood and the like because of theirchemiluminescence. Thus, 1,2 dioxetanes are eminently useful in doingmedical assays.

Generally, 1,2-dioxetanes are thermally labile substances having a widerange of stability which decompose on heating and emit light, andcorrespond to the following formula (1):

Where each R corresponds to any one of a multitude of organic moietieswidely reported in the prior art, as detailed herebelow. As noted these1,2-dioxetanes have a wide range of stability. For example, the priorart, as found in: (a) K. W. Lee, L. A. Singer and K. D. Legg, J. Org.Chem., 41, 2685(1976); (b) F. McCapra, I. Beheshti, A. Burford, R. A.Hanu and K. A. Zaklika, J. Chem. Soc., Chem. Commun., 944(1977); and (c)J. H. Wieringa, J. Strating, H. Wynberg and W. Adam, Tet. Lett., 169(1972); respectively, disclose the following 1,2-dioxetanes of differentstability:

Although these high energy compounds are all spiro-substituted1,2-dioxetanes, spiroadamantane substitution exerts a tremendousstabilizing effect on these four-membered ring peroxides. The loweractivation energy (EA) of the dioxetanes of formulae (3) and (4) aboveis explained by the donation of charge from nitrogen to the dioxetanering. The dioxetane of formula (5) above decomposes at 150° C. and has ahalf life at 25° C. more than 20 years.

A 1,2-dioxetane (6) below, dispiro[adamantane2,3-[1,2-]dioxetane-4,9-fluorene] was isolated as crystals and described by W.Adam and L. A. A. Encarnacion, Chem. Ber., 115, 2592 (1982).

The stability of 1,2-dioxetanes (5) and (6) was described on the basisof bulky and rigid spiro nature of the adamantane group.

The first stable and enzymatic triggerable 1,2-dioxetane was synthesisedby the oxidation of (6-acetoxy-2-naphthyl) methoxy methyleneadamantaneas reported by A. P Schaap, R. S. Handley and B. P Giri, Tet. lett., 935(1987). This 1,2-dioxetane utlizes aryl esterase emzyme to catalyze thecleavage of the acetate group of anaphthylacetate-substituted-1,2-dioxetane and produce chemiluminescencein aqueous buffers at ambient temperature by the following sequence:

Several other stabilized 1,2-dioxetanes and their use as enzymesubstrates have been disclosed in the literature. See, inter alia, A. P.Schaap, T. S. Chen, R. S. Handley, R. DeSilva and B. R Giri, Tet. Lett.,1155(1987); A. P. Schaap, M. D. Sandison and R. S. Handley, Tet. Lett.,1159 (1987); U.S. Pat. No. 4,962,192; U.S. Pat. No. 4,978,614; U.S. Pat.No. 5,386,017; U.S. Pat. No. 5,721,370, the disclosures of which arehereby incorporated by reference.

These several other 1,2-dioxetanes, generally, have the followinggeneral structures:

wherein

is a non-active site and which is selected from the group of polycyclicalkyl groups containing 6 to 30 carbon atoms, OX is an oxy groupsubstituted on an aryl ring which forms an unstable oxide intermediate1,2-dioxetane compound when triggered to remove X by an activating agentselected from the group consisting of an acid, a base, a salt, an enzymeand an inorganic or organic catalyst, and electron donor source, and Xis a chemically labile group which is removed by the activating agentsto form light and carbonyl containing compounds, R₁ is a lower alkylcontaining 1 to 8 carbon atoms, or mixtures thereof, or

where T is a non-active site which is a cycloalkyl or a polycycloalkylgroup bonded to the 4-membered ring portion of the dioxetane by a spirolinkage; Y is a fluorescent chromophore; X is a hydrogen, alkyl, arylarylkyl, alkaryl, heteroalkyl. heteroaryl, cycloalkyl, cycloheteroalkyl,or enzyme cleavable group; and Z is hydrogen or an enzyme cleavablegroup, provided that at least one of X or Z must be an enzyme cleavablegroup.

The enzyme cleavable 1,2-dioxetanes of formulae (14), (15) and (16)shown below have been commercialized and used in immuno assays, southernblotting, northern blotting, western blotting, plaque/colony lifts andDNA sequencing.

The 1,2-dioxetane of formula (15) with the chloro substitution in theadamantane ring demonstrates better results in DNA sequencing whencompared to dioxetane (14). Dioxetane (16) is more soluble in an aqueoussystem than 1,2-dioxetane (14) and (15) when a CH₃ group is replacedwith a CH₂CH₂CH₂COOH.

Other relevant prior art can be found in U.S. Pat. Nos. 5,386,017;4,962,192; 5,018,827; 5,578,253; 5,004,565; 5,068,339, the disclosuresof which are hereby incorporated by reference.

While these prior art compounds provide enzyme cleavable 1,2-dioxetanes,it has been observed that in an aqueous buffer, the luminescence ofthese molecules is particularity poor, especially when trace amounts ofbiological materials are sought to be detected. Thus more powerfuldioxetanes are needed i.e. dioxetanes having higher levels ofchemiluminescence in an aqueous buffer.

SUMMARY OF THE INVENTION

The present invention provides novel 1,2-dioxetanes derived fromspiro-fused ketones with or without π-electrons in the ring or withcarbon-carbon double bond(s) in the spiro-fused ring. Additionally,these new dioxetanes have electron donating or withdrawing groups at thefour-membered peroxide ring to render these dioxetanes active at allsites.

The 1,2-dioxetanes hereof generally correspond to the formula:

wherein

-   (1) when Ar—O—Y and OR join together to give an aryl group    substituted with an X-oxy group to form a stable 1,2-dioxetane    intermediate which is triggerable to form an unstable intermediate    oxide, R₂ and R₃ either form (a)

which is either a cyclic, polycyclic or spiro-fused ring containing atleast one carbon-carbon double bond or cabon-carbon triple bond in thering or side chain with or without heteroatoms or (b)

which is a cyclic, polycyclic or spiro-fused ring containing substitutedor unsubstituted fused aromatic ring or substituted or unsubstitutedaromatic ring attached by linker arms; or

-   (2) when Ar—O—Y and OR₁ do not join together

(a) Ar is aryl and may be phenyl, substituted phenyl, naphthyl,substituted naphthyl, anthryl, substituted anthryl or other aromatic ornonaromatic fluorescent or nonfluorescent group; Y is a hydrogen, alkyl,acetate, t-butyldimethylsilyl or an enzyme cleaveable group, an antibodycleaveable group; R₁ is selected from the group consisting of alkyl,aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl,cycloheteroalkyl, alkyletheralkyl, alkyletheraryl, alkyl(etheralkyl)₂,alkyl(etheralkyl)₃, alkyletherhaloalkyl, alkyl(etherhaloalkyl)₂,alkylalkene, alkylalkyne, arylalkene, arylalkyne, halogenatedalkyl(mono, di, tri or any position in normal or branched or cyclicchain), alkylalcohol, alkylnitrile, alkylamine, alkylacid (mono ordibasic) or the inorganic salts thereof, haloalkylalcohol,haloalkylnitrile, haloalkylamine, haloalkylacid (mono or dibasic) orinorganic salts, linker-flourescent molecule, linker-antibodies,linker-antigen, linker-biotin, linker-avidin, linker-protein orlinker-carbohydrates or linker-lipids; when R₂ and R₃ forms either (i)

which is a cyclic, polycyclic or spiro-fused ring containing at leastone carbon-carbon double bond or cabon-carbon triple bond in the ring orside chain with or without heteroatoms, or (ii)

which is a cyclic, polycyclic or spiro-fused ring containing substitutedor unsubstituted fused aromatic ring or substituted or unsubstitutedaromatic rings attached by linker arms, or

(b). Ar is aryl and may be phenyl, substituted phenyl, naphthyl,substituted naphthyl, anthryl, substituted anthryl or other aromatic ornonaromatic fluorescent or nonfluorescent group; Y is a hydrogen, alkyl,acetate, t-butyldimethylsilyl or an enzyme cleaveable group, or anantibody cleaveable group; R₁ is selected from the group consisting ofalkyletheralkyl, alkyletheraryl, alkyl(etheralkyl)₂, alkyl(etheralkyl)₃,alkyletherhaloalkyl, alkyl(etherhaloalkyl)₂, alkylalkene, alkylalkyne,arylalkene, arylalkyne, halogenated alkyl(mono, di, tri or any positionin normal or branched or cyclic chain), alkylalcohol, alkylnitrile,alkylamine, alkylacid (mono or dibasic) or the inorganic salts thereof,haloalkylalcohol, haloalkylnitrile, haloalkylamine, haloalkylacid (monoor dibasic) or inorganic salts, linker-flourescent molecule,linker-antibodies, linker-antigen, linker-biotin, linker-avidin,linker-protein or linker- or linker-lipids; when R₂ and R₃ form

which is a cyclic or polycyclic alkyl group or spiro-fused ring with orwithout substitution or

(c). Ar is aryl and may be phenyl, substituted phenyl, naphthyl,substituted naphthyl, anthryl, substituted anthryl or any other aromaticor nonaromatic fluorescent or nonfluorescent group; Y is a hydrogen,alkyl, acetate, t-butyldimethylsilyl or an enzyme cleaveable group, oran antibody cleaveable group; R₁ is selected from the group consistingof alkyletheralkyl, alkyletheraryl, alkyl(etheralkyl)₂,alkyl(etheralkyl)₃, alkyletherhaloalkyl, alkyl(etherhaloalkyl)₂,alkylalkene, alkylalkyne, arylalkene, arylalkyne, halogenatedalkyl(mono, di, tri or any position in normal or branched or cyclicchain), alkylalcohol, alkylnitrile, alkylamine, alkylacid (mono ordibasic) or the inorganic salts thereof, haloalkylalcohol,haloalkylnitrile, haloalkylamine, haloalkylacid (mono or dibasic) orinorganic salts, linker-flourescent molecule, linker-antibodies,linker-antigen, linker-biotin, linker-avidin, linker-protein orlinker-carbohydrates or linker-lipids; where R₂ and R₃ are branchedalkyl and cycloalkyl groups containing 3 to 8 carbon atoms which cancontain halogens and hetero atoms in the ring or side chain thereof.

The new dioxetanes are triggered by the same activating agents describedabove

The new alkenes hereof used to prepare the 1,2-dioxetanes hereof areprepared by the reaction of (a) 2-adamantanone or other spiro-fusedketone including ketones having a π-electron in the ring with (b) asubstituted aromatic ester or ketone, using titanium trichloride ortetrachloride and a reducing agent such as an active metal or lithiumaluminium hydride in tetrahydrofuran (THF) or other solvent of choice.This reaction is an intermolecular coupling of a ketone and an ester orketone to form a vinyl ether using a modified McMurray procedure.Ordinarily, the reactants are present in at least stoichiometricquantities. However, excess amounts of the ester or ketone can be used.The temperatures at which the reactions as described above are thosedisclosed in the art.

Photooxygenation of the resulting vinyl ether by well-known conventionaltechniques affords 1,2-dioxetanes that are easily handled compounds withthe desired stability.

The chemiluminescent decomposition of the 1,2-dioxetanes hereof, asnoted above, can, preferably, can be conveniently triggered at roomtemperature by removing the protecting group with a fluoride ion, baseor an enzyme to generate the unstable, aryloxide 1,2-dioxetaneintermediate which cleaves to the starting materials and yields intenseblue or other colored luminescence light.

For a more complete understanding of the present invention reference ismade to the following detailed description and accompanying examples.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As stated above chemiluminescent enzyme substrates based on,1,2-dioxetane are well known in the literature for biological assayssuch as immunoassays and DNA probes. Use of these high energy compoundsin biological systems requires 1,2-dioxetanes which are thermally stableat the temperature of the enzymatic reaction and which do not undergorapid spontaneous decomposition in an aqueous buffer. The spiro-fusedadamantyl dioxetanes hereof meet these requirements. The present1,2-dioxetanes can be modified as substrates for various enzymesincluding aryl esterase, β-galactosidase, alkaline phosphatase andothers.

In accordance herewith and as noted above, the present inventionprovides new 1,2-dioxetanes. These new 1,2-dioxetanes hereof correspondto the formula:

wherein

-   (1) when Ar—O—Y and OR join together to give an aryl group    substituted with an X-oxy group to form a stable 1,2-dioxetane    intermediate which is triggerable to form an unstable intermediate    oxide, R₂ and R₃ either form (a)

which is a cyclic, polycyclic or spiro-fused ring containing at leastone carbon-carbon double bond or cabon-carbon triple bond in the ring orside chain with or without heteroatoms or (b)

which is a cyclic, polycyclic or spiro-fused ring containing substitutedor unsubstituted fused aromatic ring or substituted or unsubstitutedaromatic ring attached by linker arms; or

-   (2) when Ar—O—Y and OR₁ do not join together

(a) Ar is aryl and may be phenyl, substituted phenyl, naphthyl,substituted naphthyl, anthryl, substituted anthryl or other aromatic ornonaromatic fluorescent or nonfluorescent group; Y is a hydrogen, alkyl,acetate, t-butyldimethylsilyl or an enzyme cleaveable group, or anantibody cleaveable group; R₁ is selected from the group consisting ofalkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl,cycloheteroalkyl, alkyletheralkyl, alkyletheraryl, alkyl(etheralkyl)₂,alkyl(etheralkyl)₃, alkyletherhaloalkyl, alkyl(etherhaloalkyl)₂,alkylalkene, alkylalkyne, arylalkene, arylalkyne, halogenatedalkyl(mono, di, tri or any position in normal or branched or cyclicchain), alkylalcohol, alkylnitrile, alkylamine, alkylacid (mono ordibasic) or the inorganic salts thereof, haloalkylalcohol,haloalkylnitrile, haloalkylamine, haloalkylacid (mono or dibasic) orinorganic salts, linker-flourescent molecule, linker-antibodies,linker-antigen, linker-biotin, linker-avidin, linker-protein orlinker-carbohydrates or linker-lipids; when R₂ and R₃ form either (i)

which is a cyclic, polycyclic or spiro-fused ring containing at leastone carbon-carbon double bond or cabon-carbon triple bond in the ring orside chain with or without heteroatoms, or (ii)

which is a cyclic, polycyclic or spiro-fused ring containing substitutedor unsubstituted fused aromatic ring or substituted or unsubstitutedaromatic ring attached by linker arms, or

(b). Ar is aryl and may be phenyl, substituted phenyl, naphthyl,substituted naphthyl, anthryl, substituted anthryl or any other aromaticor nonaromatic fluorescent or nonfluorescent group; Y is a hydrogen,alkyl, acetate, t-butyldimethylsilyl or an enzyme cleaveable group, oran antibody cleaveable group; R₁ is selected from the group consistingof alkyletheralkyl, alkyletheraryl, alkyl(etheralkyl)₂,alkyl(etheralkyl)₃, alkyletherhaloalkyl, alkyl(etherhaloalkyl)₂,alkylalkene, alkylalkyne, arylalkene, arylalkyne, halogenatedalkyl(mono, di, tri or any position in normal or branched or cyclicchain), alkylalcohol, alkylnitrile, alkylamine, alkylacid (mono ordibasic) or the inorganic salts thereof, haloalkylalcohol,haloalkylnitrile, haloalkylamine, haloalkylacid (mono or dibasic) orinorganic salts, linker-flourescent molecule, linker-antibodies,linker-antigen, linker-biotin, linker-avidin, linker-protein orlinker-carbohydrates or linker-lipids; when R₂ and R₃ form

which is a cyclic, polycyclic alkyl group with or without substitutionwhich are spiro-fused to the dioxetane ring, or

(c). Ar is aryl and may be phenyl, substituted phenyl, naphthyl,substituted naphthyl, anthryl, substituted anthryl or any other aromaticor nonaromatic fluorescent or nonfluorescent group; Y is a hydrogen,alkyl, acetate, t-butyldimethylsilyl or an enzyme cleaveable group, oran antibody cleaveable group; R₁ is selected from the group consistingof alkyletheralkyl, alkyletheraryl, alkyl(etheralkyl)₂,alkyl(etheralkyl)₃, alkyletherhaloalkyl, alkyl(etherhaloalkyl)₂,alkylalkene, alkylalkyne, arylalkene, arylalkyne, halogenatedalkyl(mono, di, tri or any position in normal or branched or cyclicchain), alkylalcohol, alkylnitrile, alkylamine, alkylacid (mono ordibasic) or the inorganic salts thereof, haloalkylalcohol,haloalkylnitrile, haloalkylamine, haloalkylacid (mono or dibasic) orinorganic salts, linker-flourescent molecule, linker-antibodies,linker-antigen, linker-biotin, linker-avidin, linker-protein orlinker-simple or complex carbohydrates or linker-simple and complexlipids; Where R₂ and R₃ are branched alkyl and cycloalkyl groupscontaining 3 to 8 carbon atoms which can contain halogens and heteroatoms in the ring or side chain thereof.

Typically, the new alkenes hereof are prepared by the reaction of (a)2-adamantanone or other spiro-fused ketone including ketones having aπ-electron in the ring with (b) a substituted aromatic ester or ketone,using titanium trichloride or tetrachloride and a reducing agent such asan active metal or lithium aluminium hydride in tetrahydrofuran (THF).This reaction is an intermolecular coupling of a ketone and an ester orketone to form a vinyl ether using a modified McMurray procedure.Ordinarily, the reactants are present in at least stoichiometricquantities. However, excess amounts of the ester or ketone can be used.The temperatures at which the reactions as described above are thosedisclosed in the art.

Photooxygenation of the resulting vinyl ether affords 1,2-dioxetanesthat are easily handled compounds with the desired stability.

When these dioxetanes react with an activating reagent or agent whichremoves the Y moiety (formula 17), they decompose to form an aryl oxide1,2-dioxetane intermediate of the formula:

This aryl oxide oxides 1,2-dioxetane intermediate, then, spontaneouslydecomposes to produce light and compounds of the formulae:

where compound (19) is the starting organic ketone and compound (20) isthe residue of the starting organic ester or ketone when Ar O⁻ and OR₁join together.

In practicing the present invention, compound (19) can be any one of ora mixture of adamantan-2-one, substituted adamantan-2-one,adamantan-2-one-4,5-ene, substituted adamantan-2-one-4,5-ene,2-hydroxytricyclo[7.3.1.0^(2,7)] tridecan-13-one or substituted2-hydroxytricyclo [7.3.1.0^(2,7)] tridecan-13-one,tricyclo[7.3.1.0^(2,7)] tridec-2,7-ene-13-one or substitutedtricyclo[7.3.1.0^(2,7)] tridec-2,7-ene-13-one, bicyclo[3.3.1]nonan-9-one or substituted bicyclo[3.3.1]nonan-9-one benzonorbonen-7-oneor substitutrd benzonorbornen-7-one, 2,4-dimethyl-3-propanone orsubstituted 2,4-dimethyl-3-propanone, dicyclopropyl ketone orsubstituted dicyclopropyl ketone, dicyclohexyl ketone or substituteddicyclohexyl ketone when compound (20) is selected from the groupconsisting of substituted or unsubstituted 9H-fluoren-9-one,9H-xanthen-9-one, 2,2,2-trifluoroethyl 3-hydroxybenzoate or substituted2,2,2-trifluoroethyl 3-hydroxybenzoate, 2-phenoxyethyl 3-hydroxybenzoateor substituted 2-phenoxyethyl 3-hydroxybenzoate, and the like furtherthe ketone of formula (19) is selected from the group consisting ofadamantan-2-one-4,5-ene, substituted adamantan-2-one-4,5-ene,tricyclo[7.3.1.0^(2,7)] tridec-2,7-ene-13-one, substitutedtricyclo[7.3.1.0^(2,7)] tridec-2,7-ene-13-one when the compound offormula (20) is alkyl or aryl 3-hydroxybenzoate or substituted alkyl oraryl 3-hydroxybenzoate.

The alkenes hereof used to prepare the present dioxetane correspond tothe formula:

wherein R₁, R₂, R₃, Y and Ar are as described above,

These alkenes are prepared by the coupling of the above-describedketones and esters or ketones.

Generally, the intramolecular coupling reaction between the ketone andthe ester or ketone is carried out at a temerature ranging from about25° C. to about 85° C. and, perferably, from about 45° C. to about 65°C. A stocichiometric excess of either the ester or ketone may be used.

The coupling reaction is carried out in the presence of suitablesolvents and active metals as described in the prior art denoted above,the disclosure above which are hereby incorporated by reference.

After the alkene is obtained it is then, photooxidized to form thestable, triggerable 1,2-dioxetane hereof. These dioxetanes can, then, bede-stablized or triggered by the reaction with base, acid, enzyme and orinorganic or organic catalyst and or electron donor source in thepresence or absence of a fluorscence compound, as described in theliterature or above cited prior art.

For a more complete understanding of the present invention, reference ismade to the following non-limiting examples. In the examples, all partsand percentages are by weight unless expressly stated to be otherwise.

In supporting the findings, the structures of the resulting compoundswas confirmed by Nuclear Magnetic Resonance (NMR). NMR spectra wererecorded on a General Electric QE 300 spectrometer in desired solventsusing tetramethylsilane as an internal standard. Chemiluminescencekinetics were performed on a Monolight 1500 at room temperature. Thepurity of the materials were checked by TLC on silica gel plate. Meltingpoints were measured in a MEL-TEMPII capillary melting point apparatusand are uncorrected. All the alkenes were dissolved in a suitablesolvent and photooxidized by irradiation with 1000-W sodium lamp underbubbled oxygen at ice-water temperature in the presence ofpolystyrene-bound Rose Benzal as reported in the literature.

EXAMPLE I

This example illustrates the preparation of[(4-Methoxy)-4-(3-phosphoryloxyphenyl)] spiro[1,2-dioxetane-3,13′-(8-n-propyl) tricyclo [7.3.1,o^(2,7)]tridec-2,7-ene], disodium salt (32) in accordance with the presentinvention. The sequence of the reactions can be shown as:

-   (a) Synthesis of    8-n-propyl-2-hydroxytricyclo[7.3.1.0^(2,7)]tridecan-13-one (24).

Into a three-neck 500 mL round bottom flask equipped with magneticstirrer and an oil bath under nitrogen was added 250 parts ofcyclohexanone. The oil bath temperature was maintained at 70-75° C. withstirring. A solution of 2.5 parts of potassium hydroxide dissolved in 25mL of absolute ethyl alcohol was added to the reaction flask in oneportion. Thirty parts of butyraldehyde was dissolved in 35 mL ofabsolute ethyl alcohol and added dropwise to the reaction flask over aperiod of 6 hours. The reaction mixture was stirred at 70-75° C. for the15 hours. After the reaction mixture was cooled to room temperature andrefrigerated for 15 hours. The inside surface of the flask was scratchedwith a glass rod to precipitate out the white material and the flask wasrefrigerated for additional 4-5 hours. The white solid material wasfiltered and washed with 50 mL of cold water and then with 50 mL coldether. The recovered solid was dried at room temperature and the yieldwas 35 parts. The solid showed a single spot on silica gel TLC plateeluted with 10% ethyl acetate/hexane. The structure of the material wasconfirmed on the basis of ¹H NMR. This reaction proceded as follows:

-   (b) Synthesis of    8-n-propyltricyclo[7.3.1.0^(2,7)]tridec-2,7-ene-13-one (25).

Into a round bottom flask equipped with heating mantle, magnetic stirrerand a water separator was charged with 150 parts of benzene, 5 parts ofthe hydroxy ketone (24) and 0.5 parts of concentrated sulfuric acid. Thereaction mixture was heated under reflux for 12 hours. Water wasseparated and the TLC of the reaction mixture on silica gel plate showedtwo new spots and disappearance of the starting material. The reactionmixture was cooled to room temperature and the benzene solution wasfiltered. The solvent was removed under reduced pressure and theso-obtained oily material was dissolved in 150 mL of methylene chloride.The methylene chloride solution was washed twice with 50 mL of water anddried on anhydrous sodium sulfate. The solvent was removed under reducedpressure and the oily material was purified on silica gel column to give3.18 parts of an oily nature compound. The structure of the material wasconfirmed on the basis of ¹H NMR. The reaction proceded in accordancewith the following equation:

-   (c) Synthesis of methyl 3-hydroxybenzoate (27).

Into a 2 L round bottom flask equipped with magnetic stirrer and heatingmantle was added 100 parts of 3-hydroxy benzoic acid (26), 1.25 L ofmethanol and 2 mL of concentrated sulfuric acid. The reaction mixturewas heated under gentle reflux for 12 hours. The solvent was evaporatedunder reduced pressure and the recovered solid material was dissolved in500 mL of ethyl acetate. The organic layer was washed with 250 mL ofwater, 250 mL of 5% aqueous solution of sodium bicarbonate and 250 mL ofwater. The ethyl acetate layer was dried over anhydrous sodium sulfateand the solvent was evaporated under reduced pressure. The oily materialwas treated with n-hexane to give a solid material which when filteredunder reduced pressure and dried at room temperature, yield 105 parts.TLC on silica gel plate showed a single spot. The structure of thecompound was confirmed on the basis of ¹H NMR. The material was preparedaccording to the following equation:

-   (d) Synthesis of methyl 3-tert-butyldimethylsiloxybenzoate (28).

Into a 250 mL round bottom flask was added 50 mL of dry dimethylformamide. Ffifteen parts of the hydroxy benzoate (27) and seventeenparts of tert-butyldimethyl silyl chloride were added to the reactionflask with stirring. Seventeen parts of Imidazole was added in portionsand stirring was continued for 10 hours. The reaction mixture wasextracted with (3×300 mL) of hexane and the hexane layer was washedtwice with 250 mL of water. The organic layer was dried on anhydroussodium sulfate and the solvent was evaporated under reduced pressure togive an oil, yield 26 parts. This material was pure enough for the nextstep of the reaction. The material was prepared according to thefollowing equation:

-   (e) Synthesis of (3-tert-butyldimethylsiloxyphenyl) methoxymethylene    8-n-propyltricyclo [7.3.1.0^(2,7)] tridec-2,7-ene (29).

Into a 500 mL three-neck flask equipped with magnetic stirrer,pressure-equalizing addition funnel and nitrogen line was charged with200 mL anhydrous THF. Twelve parts of titanium tetrachloride was addeddropwise over a period of 30 minutes. The suspension was stirred for 20minutes and 17 parts of zinc was added in small portions. The reactionmixture was heated under reflux for 3 hours and 30 parts oftriethylamine was added dropwise. After refluxing one hour a solution of4 parts of ester (28) and 3 parts of ketone (25) in 50 mL of dry THF wasadded over a period of 90 minutes and the reaction mixture was heatedfor one hour. TLC on silica gel plate of the mixture shows the presenceof the starting ester. Two and one half parts of ketone (25) in 20 mL ofdry THF was added dropwise over 60 minutes and refluxed for two hours.The mixture was cooled to room temperature, diluted with 500 mL ofhexane and decanated. The residue was washed with hexane (3×200 mL). Thecombined hexane layer was filtered and evaporated under reduced pressureto give an oily material which was purified by chromatography on silicagel column using 2.5% ethyl acetate/hexane as an eluant. The fractionswere checked by TLC on silica gel plate and the desired fractions werecombined. The solvent was evaporated under reduced pressure to give anoil, yield 5.2 parts. The structure was confirmed by ¹H NMR. Thereaction proceeded as follows:

-   (f) Synthesis of (3-Hydroxyphenyl) methoxymethylene    8-n-propyltricyclo[7.3.1.0^(2,7)] tridec-2,7-ene (30).

To a solution of 5.2 parts of alkene (29) in 150 mL of THF was added to7 parts of a (70%) tetra-n-butylammonium fluoride in 50 mL of THF over aperiod of 10 minutes and stirring was continued for the next two hours.TLC on silica gel plate showed the formation of new product. Solvent wasevaporated under reduced pressure and the oily material was dissolved in300 mL of methylene chloride and washed with 2×150 mL of water. Afterdrying over anhydrous sodium sulfate, the solvent was evaporated and theoily product was purified on silica gel column. The fractions werechecked by TLC on silica gel plate and the combined organic solvent wasevaporated, yield 3.25 parts. The structure of the product was confirmedon the basis of ¹H NMR. The reaction proceeded as follows:

-   (g) Synthesis of (3-Phosphoryloxyphenyl) methoxymethylene    8-n-propyltricyclo[7.3.1.0^(2,7)] tridec-2,7-ene, disodium salt    (31).

Into a 500 mL three-neck round bottom flask equipped with magneticstirrer was added 60 mL of methylene chloride and 20 parts of triethylamine under nitrogen at ice-water temperature. The reaction flask wascooled to ice-water temperature and 3.5 mL of phosphorous oxychloridewas added to the reaction mixture over a period of 15 minutes. Asolution of 3.5 parts of alkene (30) in 35 mL of methylene chloride wasadded dropwise over 30 minutes and was stirred for 2 hours. The solventwas evaporated under reduced pressure. The reaction mixture wasextracted with 2×250 mL of hexanes containing 0.1% triethyl amine.Hexane was evaporated under reduced pressure and the product wasdissolved in 50 mL of acetonitrile. A solution of 2.5 parts of sodiumhydroxide in 25 mL of water was added dropwise and was stirred for twohours. Reaction mixture was diluted with 50 mL of acetonitrile and thesolid was filtered. The solid was crystalized with methanol and acetonemixture. The solid was filtered, washed with acetone and dried, yield2.9 arts. The reaction proceeded as follows:

-   (h) Photooxidation of (3-Phosphoryloxyphenyl) methoxymethylene    8-n-propyltricyclo[7.3.1.0^(2,7)] tridec-2,7-ene, disodium salt    (31).

Alkene (31) was photooxidized by the above noted procedure, to give[(4-methoxy)-4-(3-phosphoryloxyphenyl)] spiro[1,2-dioxetane-3,13′-(8-n-propyl) tricyclo[7.3.1.0^(2,7)]tridec-2,7-ene]. disodium salt (32).

EXAMPLE II

This example illustrates the preparation of[(4-Methoxy)-4-(3-phosphoryloxyphenyl)] spiro[1,2-dioxetane-3,13′-tricyclo [7.3.1,o^(2,7)]tridec-2,7-ene], disodiumsalt (39).This dioxetane was prepared by the sequence of the reactionsin accordance herewith:

-   (a) Synthesis of 2-Hydroxytricyclo[7.3.1.0^(2,7)]tridecane-13-one    (34).

Into a 500 mL three-neck round bottom flask equipped with magneticstirrer and oil bath was added 250 parts of cyclohexanone (22) undernitrogen. The oil bath temperature was maintained at 80-85° C. withstirring. Sixty mL of absolute ethanol with 2.5 parts of potassiumhydroxide was added in one portion. Sixteen parts of paraformaldehydewas added in small portions over a period of 3 hours and the temperaturewas maintained for 15 hours with stirring. The reaction mixture wascooled to room temperature and refrigerated at 4° C. for 15 hours. Theinside surface of the flask and liquid was scratched with a glass rod.The solid started to separate and the flask was refrigerated for anadditional 24 hours. The recovered solid was filtered under reducedpressure. The solid material was washed with 50 mL of water and twotimes with 50 mL of cold ether and dried, yield of 29 parts. The motherliquor was stored at −20° C. for 15 hours. The solid material wasfiltered and washed with water and ether, yield 10.0 parts. The combinedsolid was washed with hexane. The material showed a single spot on TLCon silica gel plate. The structure was confirmed on the basis of ¹H NMR.The reaction proceeded as follows:

-   (b) Synthesis of tricyclo[7.3.1.0^(2,7)]tridec-2,7-ene-13-one (35):

Into a 500 mL round bottom flask was added 5.0 parts of the hydroxyketone (34). The product was dissolved in 150 mL of benzene by heatingat 40° C. The reaction mixture was stirred at refluxing temperature for12 hours in the presence of 0.5 parts of concentrated sulfuric acid. Thereaction mixture was cooled to room temperature and the benzene solutionwas filtered. The solvent was evaporated under reduced pressure and theoily material dissolved in 150 mL of methylene chloride. The organiclayer was washed with water and dried on anhydrous sodium sulfate. Thesolvent was evaporated under reduced pressure and purified on silica gelcolumn using 10% ethyl acetate/hexanes. The desired fractions werecombined and the solvent was evaporated to give an oil, yield 3.5 parts.¹H NMR confirmed the following structure. The reaction proceeded asfollows:

-   (c) Synthesis of (3-tert-butyldimethylsiloxyphenyl) methoxymethylene    tricyclo[7.3.1.0^(2,7)] tridec-2,7-ene (36).

Into a 500 mL three-neck flask equipped with magnetic stirrer,pressure-equalizing addition funnel and nitrogen line was charged with200 mL of anhydrous THF. Twelve parts of titanium tetrachloride wasadded dropwise over a period of 30 minutes. The suspension was stirredfor 20 minutes and 17 parts of zinc was added in small portions. Thereaction mixture was heated under reflux for 2 hours and 30 mL oftriethylamine was added dropwise. After refluxing one hour, 4 parts of asolution of methyl 3-tert-butyldimethylsiloxybenzoate and 2 parts ofketone (35) in 35 mL of dry THF was added over a period of 60 minutesand the reaction mixture was heated for one hour. The mixture on TLCsilia gel plate showed the presence of the starting ester. An additional3.3 parts of starting ketone (35) in 35 mL of dry THF was added dropwiseover 90 minutes and refluxed for two hours. The mixture was cooled toroom temperature, diluted with 500 mL of hexane and decanted. Theresidue was washed with 3×200 mL of hexane. The combined hexane layerwas filtered and evaporated under reduced pressure to give an oilymaterial which was purified by chromatography on silica gel column using2.5% ethyl acetate/hexane as an eluant. The fractions were checked byTLC on silica gel plate and the desired fractions were combined. Thesolvent was evaporated under reduced pressure to give an oil, yield 4.75parts. The structure was confirmed by ¹H NMR. The reaction proceeded asfollows:

-   (d) Synthesis of (3-hydroxyphenyl) methoxymethylene    tricyclo[7.3.1.0^(2,7)] tridec-2,7-ene (37).

To a crude solution of 10 parts of (3-tert-Butyldimethylsiloxyphenyl)methoxymethylene tricyclo[7.3.1.0^(2,7)] tridec-2,7-ene in 150 mL of THFwas added 7 parts of 70% tetra-n-butylammonium fluoride in 50 mL of THFover a period of 10 minutes and stirring was continued for two hours.TLC on silica gel plate showed the formation of new product. Solvent wasevaporated under reduced pressure and the oily material was dissolved in300 mL of methylene chloride and washed with 2×150 mL of water. Afterdrying over anhydrous sodium sulfate, the solvent was evaporated and theoily product was purified on silica gel column. TLC on silica gel plateof the fractions were checked and the combined organic solvent wasevaporated, yield 3.60 parts. The structure of the product was confirmedon the basis of ¹H NMR. The reaction proceeded as follows:

-   (e) Synthesis of (3-phosphoryloxyphenyl) methoxymethylene tricyclo    [7.3.1.0^(2,7)] tridec-2,7-ene, disodium salt (38).

Into a 250 mL three-neck round bottom flask equipped with mageneticstirrer was added 15 mL of dry THF under nitrogen and then 1 mL ofphosphorous oxychloride was added drop-wise (reaction flask was cooledin ice-water bath). A solution of 1 part of alkene (37) in 15 mL of THFcontaining 0.325 parts of anhydrous pyridine was added to the reactionflask over a period of 30 minutes. The reaction mixture was stirred atroom temperature for 3 hours. TLC on silica gel plate showed theformation of new product. The solvent was evaporated to dryness and 10mL of THF was added to the reaction flask. A solution containing 0.55parts of 3-hydroxypropionitrile and 0.65 parts of anhydrous pyridine in7.5 ml of dry THF was added dropwise to the reaction mixture over 25minutes and was stirred for 15 hours at room temperature. The reactionwas cooled to ice-water temperature and the solid was filtered andwashed with cold THF. The solvent was evaporated and oily material waschromatographed on silica gel column using 70% ethyl acetate/hexanecontaining 0.1% triethyl amine. Fractions were checked by TLC on silicagel plate and the desired fractions were combined and evaporated underreduced pressure to give an oil, yield 1.20 parts. The oily material wasdissolved in 15 mL of dry THF and a solution of 0.75 parts of sodiumhydroxide in 5 mL of water was added drowise. Stirring was continued fortwo hours and the reaction mixture was diluted with 10 mL ofacetonitrile. The solid was filtered and washed with acetonitrile. Thesoild material was crystallised with methanol and acetone mixture. Thesolid was filtered and washed with acetone and dried, to yield 0.85parts. The structure was confirmed on the basis of ¹H NMR. The reactionproceeded as follows:

-   (f) Photooxidation of (3-phosphoryloxyphenyl) methoxymethylene    tricyclo [7.3.1.0^(2,7)] tridec-2,7-ene, disodium salt (38).

Alkene (38) was photooxidized as reported above, to give[(4-Methoxy)-4-(3-phosphoryloxyphenyl)] spiro[1,2-dioxetane-3,13′-tricyclo [7.3.1,o^(2,7)] tridec-2,7-ene], disodiumsalt (39). (39)

EXAMPLE III

This example illustrates the preparation of[(4-methoxy)-4-(3-phosphoryloxyphenyl] spiro[1,2-dioxetane-3,2′-adamantan-4,5-ene], disodium salt (46). The sequenceof the reactions in accordance herewith:

-   (a) Synthesis of 5-Hydroxyadamantan-2-one (40).

Into a single neck 500 mL round bottom flask equipped with a magneticstirrer and water bath, 75 parts of acetic acid containing 5 partsacetic anhydride was added. Twenty five parts of chromium trioxide wasadded in portions in 40 minutes while the temperature was maintained at15-20° C. with water bath. Five parts of Adamantan-2-one was added inportions over a period of 15 minutes. Stirring was continued for onehour. The viscous reaction mixture was poured into cold 250 mL ofaqueous 20% sodium hydroxide solution. The aqueous layer was extractedwith 3×250 mL of ethyl acetate and washed with 2×250 mL of water anddried over sodium sulfate. Solvent was evaporated under reduced pressureand chromatographed on silica gel column using 75% ethyl acetate/hexane.The desired fractions were collected and solvent was evaporated to givea solid, yield 2.6 parts, single spot on silica gel TLC plate. Thestructure was confirmed on the basis of ¹H NMR. The reaction proceededas follows:

-   (b) Synthesis of adamantan-4,5-ene-2-one (42).

Into a 500 mL round bottom flask equipped with magnetic stirrer, DeanStark water separator and a condenser, was added 150 mL of benzene and5.5 parts of hydroxy ketone (41). The solid was dissolved by heating theflask at 40° C. and 0.5 parts of concentrated sulfric acid was added.The reaction mixture was refluxed for 18 hours. After cooling to roomtemperature the solvent was evaporated under reduced pressure and theoily material was dissolved in 200 mL of ethyl acetate. The organiclayer was washed with water. The ethyl acetate layer was dried oversodium sulfate and evaporated under reduced pressure. The oily materialwas chromatographed on silica gel column using 7% ethyl acetate/hexane.The desired fractions were combined and the solvent was evaporated underreduced pressure to give an oil which on standing at 20° C., solidified,yield 4.5 parts. The structure of the product was confirmed on the basisof ¹H NMR. The reaction proceeded as follows:

-   (c) Synthesis of (3-tert-butyldimethylsiloxyphenyl) methoxymethylene    adamantan-4,5-ene (43):

Into a 500 mL three-necked flask equipped with magnetic stirrer,pressure-equalizing addition funnel and nitrogen line was charged with200 mL of anhydrous THF. Fifteen parts of titanium tetrachloride wasadded dropwise over a period of 30 minutes. The suspension was stirredfor 20 minutes and 22 parts of zinc was added in small portions. Thereaction mixture was heated under reflux for 2 hours and 37 parts oftriethylamine was added dropwise. After refluxing one hour, four partsof a solution of ester (28) and 3 parts of alkene (42) in 50 mL of dryTHF was added over a period of 90 minutes and the reaction mixture washeated for one hour. Silica gel TLC plate analysis of the mixture showedthe presence of starting ester (28). Additional 1.5 parts of startingketone (42) in 15 mL of dry THF was added dropwise over 30 minutes andrefluxed for two hours. The mixture was cooled to room temperature,diluted with 500 mL of hexane and decanated. The residue was washed with3×200 mL of hexane. The combined hexane layer was filtered andevaporated under reduced pressure to give an oily material which waspurified by chromatography on silica gel column using 2.5% ethylacetate/hexane as an eluant. The fractions were checked by TLC on silicagel plate and the desired fractions were combined. The solvent wasevaporated under reduced pressure to give an oil, yield 5.75 parts. Thestructure was confirmed by ¹H NMR. The reaction proceeded as follows:

-   (d) Synthesis of (3-Hydroxyphenyl) methoxymethylene    adamantan-4,5-ene (44).

A solution of 5.75 parts of the alkene (43) and 100 mL of THF was addedto 7.0 parts of 70% of tetra-n-butylammonium fluoride in 50 mL of THFover a period of 15 minutes. Sirring was continued for two hours. TLC onsilica gel plate showed the formation of new product. Solvent wasevaporated under reduced pressure and the oily material was dissolved in250 mL of methylene chloride and washed with 2×250 mL of water. Afterdrying over anhydrous sodium sulfate, the solvent was evaporated and theoily product was purified on silica gel column. TLC on silica gel plateof the fractions were checked and the combined organic solvent wasevaporated, yield 3.20 parts. The structure of the product was confirmedon the basis of ¹H NMR. The reaction proceeded as follows:

-   (e) Synthesis of (3-phosphoryloxyphenyl) methoxymethylene    adamantan-4,5-ene, disodium salt (45).

Thirty parts of dry THF under nitrogen and 4.0 mL of phosphorousoxychloride was added dropwise to a 250 mL three-neck round bottom flaskequipped with magenetic stirrer (reaction flask was cooled in anice-water bath). A solution of 2.0 parts of alkene (44) in 25 mL of THFcontaining 0.725 parts of anhydrous pyridine was added to the reactionflask over a period of 30 minutes and stirring was continued at roomtemperature for 3 hours. TLC on silica gel plate showed the formation ofnew product. The solvent was evaporated to dryness and 20 mL of THF wasadded to the reaction flask. A solution containing 1.55 parts of3-hydroxypropionitrile and 1.65 parts of anhydrous pyridine in 15 mL ofdry THF was added to the reaction mixture dropwise over 25 minutes andstirring was continued for 15 hours at room temperature. The reactionwas cooled to ice-water temperature and the solid was filtered, washedwith cold THF. The solvent was evaporated and resulting oily materialwas chromatographed on silica gel column using 70% ethyl acetate/hexanecontaining 0.1% triethylamine. Fractions were checked by TLC on silicagel plate and desired fractions were combined and evaporated underreduced pressure to give an oil, yield 2.50 parts. The oily material wasdissolved in 15 mL of dry THF and a solution of 1.75 parts of sodiumhydroxide in 15 mL of water was added dropwise. Stirring was continuedfor two hours and the reaction mixture diluted with 20 mL ofacetonitrile. The solid was filtered and washed with acetonitrile. Thesolid material was crystalized with methanol/acetone mixture. The solidwas filtered and washed with acetone and dried, yield, 1.95 parts. Thestructure was confirmed on the basis of ¹H NMR. The reaction proceededas follows:

-   (f) Photooxidation of (3-phosphoryloxyphenyl) methoxymethylene    adamantan-4,5-ene, disodium salt (45).

Alkene (45) was photooxidized as reported above, to give[(4-methoxy)-4-(3-phosphoryloxyphenyl]spiro[1,2-dioxetane-3,2′-adamantan-4,5-ene],disodium salt (46).

EXAMPLE IV

This example illustrates the preparation of[4-(2,2,2-trifluoroethoxy)-4-(3-phosphoryloxyphenyl)]spiro[1,2-dioxetane-3,13′-tricyclo[7.3.1.0^(2,7)]tridec-2,7-ene],disodium salt (52). The sequence of the reactions in accordanceherewith:

-   (a) Synthesis of 2,2,2-trifluoroethyl 3-hydroxybenzoate (47).

Into a 250 mL round bottom flask equipped with magnetic stirrer andheating mantle was added 10 parts of 3-hydroxy benzoic acid, 100 partsof 2,2,2-trifluoroethanol and 0.5 parts of concetrated sulfuric acid.The reaction mixture was heated at 95° C. for 48 hours. The solvent wasevaporated under reduced pressure and the solid material was dissolvedin 250 mL of ethyl acetate. The organic layer was washed with 250 mL ofwater, 250 mL 5% solution of sodium bicarbonate in 100 mL of water andfinally with 250 mL of water. The ethyl acetate layer was dried overanhydrous sodium sulfate and the solvent was evaporated under reducedpressure, yield 4.1 parts. TLC on silica gel plate showed a single spot.The structure was confirmed on the basis of ¹H NMR. The reactionproceeded as follows:

-   (b) Synthesis of 2,2,2-Trifluoroethyl    3-tert-butyldimethylsiloxybenzoate (48)

Into a 250 mL round bottom flask was added 30 mL of dry dimethylformamide and 4.0 parts of ester (47) and 3.5 parts oftert-butyldimethyl silyl chloride were added to the reaction flask withstirring. Three parts of Imidazole was added in portions and stirringwas continued for 10 hours. The reaction mixture was diluted with 80 mLof water and the product was extracted with ethyl acetate. The organiclayer was washed with water and dried on anhydrous sodium sulfate. Thesolvent was evaporated under reduced pressure to give an oil, yield 5.65parts. The structure was confirmed on the basis of ¹H NMR. The reactionproceeded as follows:

-   (c) Synthesis of (3-tert-Butyldimethylsiloxyphenyl)    (2,2,2-trifluoroethoxy) methylene]    tricyclo[7.3.1.0^(2,7)]tridec-2,7-ene (49).

Into a 500 mL three-neck flask equipped with magnetic stirrer,pressure-equalizing addition funnel and nitrogen line was charged with200 parts of anhydrous THF. Ttitanium tetrachloride (20 mL) was addeddropwise over a period of 30 minutes. The suspension was stirred for 20minutes and 30 parts of zinc was added in small portions The reactionmixture was heated under reflux for 2 hours and 50 parts oftriethylamine was added dropwise. After refluxing one hour, 5.5 parts ofa solution of ester (48) and 3.5 parts of ketone (35) in 40 mL of dryTHF was added over a period of 90 minutes and the reaction mixture washeated for one hour. TLC on silica gel plate of the mixture showed thepresence of starting ester. Two parts ketone (35) in 15 mL of dry THFwas added dropwise over 30 minutes and refluxed for two hours. Themixture was cooled to room temperature, diluted with 500 mL of hexaneand decanated. The residue was washed with 3×200 mL of hexane. Thecombined hexane layer was filtered and evaporated under reduced pressureto give an oily material which was purified by chromatography on silicagel column using 10% ethyl acetate/hexane mixture as an eluant. Thefractions were checked by TLC on silica gel plate and the desiredfractions were combined. The solvent was evaporated under reducedpressure to give an oil, yield 6.1 parts. The structure was confirmed by¹H NMR. The reaction proceeded as follows:

-   (d) Synthesis of [(3-Hydroxyphenyl) (2,2,2-trifluoroethoxy)    methylene] tricyclo[7.3.1.0^(2,7)]tridec-2,7-ene (50).

To a solution 6.1 parts of alkene (49) in 100 mL of THF was added to 7.0parts of 70% of tetra-n-butylammonium fluoridein 50 mL of THF over aperiod of 15 minutes and stirring was continued for two hours. TLC onsilica gel plate showed the formation of new product. The solvent wasevaporated under reduced pressure and the oily material was dissolved in300 mL of ethyl acetate and washed with 2×200 mL of water. After dryingover anhydrous sodium sulfate, the solvent was evaporated and the oilyproduct was purified on silica gel column. TLC on silica gel plate ofthe fractions were checked and the combined organic solvent wasevaporated, yield 3.9 parts. The structure of the product was confirmedon the basis of ¹H NMR. The reaction proceeded as follows:

-   (e) Synthesis of [(3-Phosphoryloxyphenyl) (2,2,2-trifluoroethoxy)    methylene] tricyclo[7.3.1.0^(2,7)]tridec-2,7-ene, disodium salt (51)

Into a 100 mL three-neck flask equipped with magnetic stirrer, undernitrogen was added 35 mL of methylene chloride and 2.75 parts ofphosphorous oxychloride. The flask was cooled in an ice-water bath and1.57 parts of pyridine added dropwise over a period of 20 minutes.Stirring was continued for 20 minutes and 3.5 parts of alkene (50) wasdissolved in 35 mL of methylene chloride and added to the flask over aperiod of 30 minutes. The reaction mixture was stirred for three hoursat room temperature and 3.56 parts of 2-cyanoethanol and 3.56 parts ofpyridine in 35 mL of methylene chloride was added to the reaction flask.The reaction mixture was stirred for 48 hours. The solvent wasevaporated and diluted with 150 mL of ethyl acetate. The solid wasfiltered and washed with 25 ml of ethyl acetate. The combined organiclayer was washed with water and dried over sodium sulfate. The oilymaterial was dissolved in 300 parts of acetone and 4 parts of sodiumhydroxide in 6 parts of water was added dropwise. The solid was filteredand crystalized with methanol and a mixtue of ethyl acetate and ether togive 2.9 parts of white solid. The structure of the product wasconfirmed on the basis of ¹H NMR. The reaction proceeded as follows:

-   (f) Photooxidation of [(3-Phosphoryloxyphenyl)    (2,2,2-trifluoroethoxy) methylene]    tricyclo[7.3.1.0^(2,7)]tridec-2,7-ene, disodium salt (51).

Alkene (51) was photooxidized as reported above, to give[4-(2,2,2-trifluoroethoxy)-4-(3-phosphoryloxypheny)] spiro[1,2-dioxetane-3,2′-adamantane], disodium salt (52).

EXAMPLE V

This example illustrates the preparation of[4-(2-phenoxyethoxyl)-4-(3-phosphoryloxyphenyl) spiro[1,2-dioxetane-3,2′-adamantane], disodium salt (58). The sequence of thereactions in accordance herewith:

-   (a) Synthesis of 2-Phenoxyethyl 3-hydroxybenzoate (53).

Into a 250 mL round bottom flask equipped with magnetic stirrer andheating mantle was added 16.0 parts of 3-hydroxy benzoic acid, 100 partsof 2-phenoxyethanol and 0.5 parts of concentrated sulfuric acid. Thereaction mixture was heated at 95° C. for 48 hours. The excess of2-phenoxyethanol was evaporated under reduced pressure at 90° C. Thesolid material was dissolved in 250 mL of ethyl acetate. The organiclayer was first washed with 200 mL of water, second with 250 mL of 5%solution of sodium bicarbonate in water and finally washed with 3×250 mLof water. The ethyl acetate layer was dried over anhydrous sodiumsulfate and the solvent was evaporated under reduced pressure. Thismaterial was purified by chromatography on silica gel using 20% ethylacetate/hexane. The desired fractions were combined and solvent wasevaporated to give an oily material which on treatment with hexane gavea solid, yield 5.1 parts. TLC on silica gel plate showed single spot.The structure of the product was confirmed on the basis of ¹H NMR. Thereaction proceeded as follows:

-   (b) Synthesis of 2-phenoxyethyl 3-tert-butyldimethylsiloxybenzoate    (54)

Into a 250 mL round bottom flask was added 30 mL of dry dimethylformamide. Five parts of hydroxy ester (53) and 3.5 parts oftert-butyldimethyl silyl chloride were added to the reaction flask withstirring. Three parts of Imidazole was added in portions and stirringwas continued for 10 hours. The reaction mixture was diluted with 80 mLof water and the product was extracted with ethyl acetate. The organiclayer was washed with water and dried on anhydrous sodium sulfate. Thesolvent was evaporated under reduced pressure to give an oil, yield 6.80parts. This material was pure enough for next step of the reaction. Thereaction proceeded as follows:

-   (c) Synthesis of [(3-tert-Butyldimethylsiloxyphenyl)    (2-phenoxyethoxy) methylene] adamantane (55).

Into a 500 mL three-neck flask equipped with magnetic stirrer,pressure-equalizing addition funnel and nitrogen line was charged with200 mL anhydrous THF. Fifteen parts of titanium tetrachloride was addeddropwise over a period of 30 minutes. After stirring the suspension for20 minutes, 22 parts of zinc was added in small portions. The reactionmixture was heated under reflux for 2 hours and 37 mL of triethylaminewas added dropwise. After refluxing one hour a solution 5.9 parts ofalkene (54) and 3.0 parts of adamantan-2-one in 40 mL of dry THF wasadded over a period of 90 minutes. The reaction mixture was heated forone hour. TLC on silica gel plate of the mixture showed the presence ofstarting ester. One and a half part of Adamantan-2-one in 20 mL of dryTHF was added dropwise over 30 minutes and refluxed for two hours. Themixture was cooled to room temperature, diluted with 500 mL of hexaneand decanated. The residue was washed three times with 200 mL of hexane.The combined hexane layer was filtered and evaporated under reducedpressure to give an oily material which was purified by chromatographyon silica gel column using 2.5% ethyl acetate/hexane as an eluant. Thefractions were checked by TLC on silica gel plate and the desiredfractions were combined. The solvent was evaporated under reducedpressure to give an oil, yield 6.30 parts. The structure was confirmedby ¹H NMR. The reaction proceeded as follows:

-   (d) Synthesis of [(3-Hydroxyphenyl) (2-phenoxyethoxy) methylene]    adamantane (56).

To a solution of 6.30 parts of alkene (55) in 100 mL of THF was added7.0 parts of 70% mixture of tetra-n-butylammonium fluoride in 50 mL ofTHF over a period of 15 minutes. The reaction mixture was stirred fortwo hours. TLC on silica gel plate showed the formation of new product.The solvent was evaporated under reduced pressure and the oily materialwas dissolved in 300 mL of ethyl acetate and washed with two times with200 mL of water. After drying over anhydrous sodium sulfate, the solventwas evaporated and the oily product was purified on silica gel column.TLC on silica gel plate of the fractions were checked and the combinedorganic solvent was evaporated, yield 3.75 parts. The structure of theproduct was confirmed on the basis of ¹H NMR. The reaction proceeded asfollows:

-   (e) Synthesis of [(3-Phosphoryloxyphenyl) (2-phenoxyethoxy)    methylene] adamantane, disodium salt (57).    Into a 100 mL three-neck flask equipped with magnetic stirred under    nitrogen was added 10 mL of anhydrous pyridine. The flask was cooled    in a ice-water bath and 4 mL of phosphorous oxychloride was added    dropwise over a period of 20 minutes. The reaction mixture was    stirred for 20 minutes. Two parts of alkene (56) was dissolved in 10    parts of pyridine and added to the flask over a period of 30    minutes. The reaction mixture was stirred for two hours at room    temperature. This material was poured carefully into 15.0 parts of    sodium hydroxide in 50 mL of water containing 70.0 g of crushed ice.    The stirring was continued for 15 hours. Solid was filtered and    washed with acetone. The solid material was crystalized with    methanol and acetone, yield 1.35 parts. The reaction proceeded as    follows:

-   (f) Photooxidation of [(3-Phosphoryloxyphenyl) (2-phenoxyethoxy)    methylene] adamantane, disodium salt (57).

Alkene (57) was photooxidized as reported above, to give[4-(2-phenoxyethoxyl)-4-(3-phosphoryloxyphenyl)] spiro[1,2-dioxetane-3,2′-adamantane], disodium salt (58).

EXAMPLE VI

This example illustrates the preparation of[4-(2-phenoxyethoxyl)-4-(3-phosphoryloxyphenyl)spiro [1,2-dioxetane-313′-tricyclo[7.3.1.0^(2,7)]tridec-2,7-ene], disodium salt (62). Thesequence of the reactions in accordance herewith:

-   (a) Synthesis of    [(3-tert-Butyldimethylsiloxyphenyl)(2-phenoxyethoxy)methylene]tricyclo[7.3.1.0^(2,7)]tridec-2,7-ene    (59).

Into a 1000 mL three-neck flask equipped with magnetic stirrer,pressure-equalizing addition funnel and nitrogen line was charged with300 mL anhydrous THF. Twenty two parts of titanium tetrachloride wasadded dropwise over a period of 30 minutes. After stirring thesuspension for 20 minutes, 34 parts of zinc was added in small portions.The reaction mixture was heated under reflux for 2 hours and 57 mL oftriethylamine was added dropwise. After refluxing one hour a solution 10parts of ester (54) and 6.2 parts of ketone (35) in 60 mL of dry THF wasadded over a period of 60 minutes. The reaction mixture was heated forone hour. TLC on silica gel plate of the mixture showed the presence ofstarting ester. Two and a half parts of ketone (35) in 25 mL of dry THFwas added dropwise over 40 minutes and refluxed for two hours. Themixture was cooled to room temperature, diluted with 1000 mL of hexaneand decanated. The residue was washed three times with 250 mL of hexane.The combined hexane layer was filtered and evaporated under reducedpressure to give an oily material which was purified by chromatographyon silica gel column using 10% ethyl acetate/hexane as an eluant. Thefractions were checked by TLC on silica gel plate and the desiredfractions were combined. The solvent was evaporated under reducedpressure to give an oil, yield 10.8 parts. Silica gel TLC shows singlespot. The reaction proceeded as follows:

-   (b) Synthesis of [(3-Hydroxyyphenyl) (2-phenoxyethoxy) methylene]    tricyclo[7.3.1.0^(2,7)]tridec-2,7-ene (60).    To a solution of 10.8 parts of alkene (59) in 150 mL of THF was    added 10 parts of 70% mixture of tetra-n-butylammonium fluoride in    60 mL of THF over a period of 15 minutes. The reaction mixture was    stirred for two hours. TLC on silica gel plate showed the formation    of new product. The solvent was evaporated under reduced pressure    and the oily material was dissolved in 350 mL of ethyl acetate and    washed with two times with 200 mL of water. After drying over    anhydrous sodium sulfate, the solvent was evaporated and the oily    product was purified on silica gel column. TLC on silica gel plate    of the fractions were checked and the combined organic solvent was    evaporated, to yield 6.0 parts. The structure of the product was    confirmed on the basis of ¹H NMR. The reaction proceeded as follows:

-   (c) Synthesis of [(3-Phosphoryloxyphenyl) (2-phenoxyethoxy)    methylene] tricyclo[7.3.1.0^(2,7)]tridec-2,7-ene, disodium salt    (61).

Into a 100 mL three-neck flask equipped with magnetic stirrer, undernitrogen was added 45 mL of methylene chloride and 4.57 parts ofphosphorous oxychloride. The flask was cooled in an ice-water bath and2.607 parts of pyridine in 45 mL of methylene chloride added dropwiseover a period of 20 minutes. Stirring was continued for 20 minutes and6.0 parts of alkene (60) was dissolved in 45 mL of methylene chlorideand added to the flask over a period of 30 minutes. The reaction mixturewas stirred for three hours at room temperature and 5.94 parts of2-cyanoethanol and 5.94 parts of pyridine in 45 mL of methylene chloridewas added to the reaction flask. The reaction mixture was stirred for 48hours. The solvent was evaporated and diluted with 250 mL of ethylacetate. The solid was filtered and washed with 50 ml of ethyl acetate.The combined organic layer was washed with water and dried over sodiumsulfate. The oily material was dissolved in 600 parts of acetone and 8parts of sodium hydroxide in 12.5 parts of water was added dropwise. Thesolid was filtered and crystalized with methanol and a mixture of ethylacetate and ether to give 7.5 parts of white solid. The structure of theproduct was confirmed on the basis of ¹H NMR. The reaction proceeded asfollows:

-   (d) Photooxidation of [(3-Phosphoryloxyphenyl) (2-phenoxyethoxy)    methylene] tricyclo[7.3.1.0^(2,7)]tridec-2,7-ene, disodium salt    (61).

Alkene (61) was photooxidized as reported above, to give[4-(2-phenoxyethoxyl)-4-(3-phosphoryloxyphenyl)] spiro [1,2-dioxetane-313-tricyclo[7.3.1.0^(2,7)]tridec-2,7-ene], disodium salt (62).

EXAMPLE VII

This example illustrates the preparation of[4-(2-phenoxyethoxyl)-4-(3-phosphoryloxyphenyl) spiro[1,2-dioxetane-3,2′-5-chloro-adamantane], disodium salt (58). Thesequence of the reactions in accordance herewith:

-   (a) Synthesis of 5-chloroadamantan-2-one (63).

In a round bottom flask 6 parts of 5-hydroxyadamantan-2-one wasdissolved in 36 mL of thionylchloride and boiled under refluxed for twohours. The excess thionyl chloride was evaporated. The residue wasdissolved in 100 mL of methylene chloride and washed with 0.5N sodiumhydroxide and then 10% aqueous sodium chloride solution. The organiclayer was dried on sodium sulfate and the solvent evaporated underreduced pressure to give a white solid, yield 6.7 g. The reactionproceed as follows:

-   (b) Synthesis of [(3-tert-Butyldimethylsiloxyphenyl)    (2-phenoxyethoxy) methylene]-5-chloroadamantane (64).

Into a 500 mL three-neck flask equipped with magnetic stirrer,pressure-equalizing addition funnel and nitrogen line was charged with150 mL anhydrous THF. Titanium tetrachloride (12 mL) was added dropwiseover a period of 30 minutes. After stirring the suspension for 20minutes, 18 parts of zinc was added in small portions. The reactionmixture was heated under reflux for 2 hours and 29 mL of triethylaminewas added dropwise. After refluxing one hour a solution 5.0 parts ofester (54) and 3.0 parts of 5-chloroadamantan-2-one in 35 mL of dry THFwas added over a period of 45 minutes. The reaction mixture was heatedfor one hour. TLC on silica gel plate of the mixture showed the presenceof starting ester. One part of 5-chloroadamantan-2-one in 20 mL of dryTHF was added dropwise over 30 minutes and refluxed for two hours. Themixture was cooled to room temperature, diluted with 400 mL of hexaneand decanated. The residue was washed three times with 150 mL of hexane.The combined hexane layer was filtered and evaporated under reducedpressure to give an oily material which was purified by chromatographyon silica gel column using 5% ethyl acetate/hexane as an eluant. Thefractions were checked by TLC on silica gel plate and the desiredfractions were combined. The solvent was evaporated under reducedpressure to give an oil, yield 4.9 parts. The structure was confirmed by¹H NMR. The reaction proceeded as follows:

-   (c) Synthesis of [(3-Hydroxyphenyl) (2-phenoxyethoxy)    methylene]-5-chloroadamantane (65).

To a solution of 4.9 parts of alkene (64) in 75 mL of THF was added 5.0parts of 70% mixture of tetra-n-butylammonium fluoride in 25 mL of THFover a period of 15 minutes. The reaction mixture was stirred for twohours. TLC on silica gel plate showed the formation of new product. Thesolvent was evaporated under reduced pressure and the oily material wasdissolved in 150 mL of ethyl acetate and washed with two times with 200mL of water. After drying over anhydrous sodium sulfate, the solvent wasevaporated and the oily product was purified on silica gel column. TLCon silica gel plate of the fractions were checked and the combinedorganic solvent was evaporated, yield 2.5 parts. The structure of theproduct was confirmed on the basis of ¹H NMR. The reaction proceeded asfollows:

-   (d) Synthesis of [(3-Phosphoryloxyphenyl) (2-phenoxyethoxy)    methylene]-5-chloroadamantane, disodium salt (66).

Into a 100 mL three-neck flask equipped with magnetic stirrer, undernitrogen was added 25 mL of methylene chloride and 1.53 parts ofphosphorous oxychloride. The flask was cooled in an ice-water bath and0.87 parts of pyridine in 10 mL of methylene chloride added dropwiseover a period of 10 minutes. Stirring was continued for 20 minutes and 6parts of alkene (65) was dissolved in 25 mL of methylene chloride andadded to the flask over a period of 30 minutes. The reaction mixture wasstirred for three hours at room temperature and 2.0 parts of2-cyanoethanol and 2.0 parts of pyridine in 25 mL of methylene chloridewas added to the reaction flask. The reaction mixture was stirred for 48hours. The solvent was evaporated and diluted with 100 mL of ethylacetate. The solid was filtered and washed with 50 ml of ethyl acetate.The combined organic layer was washed with water and dried over sodiumsulfate. The oily material was dissolved in 150 parts of acetone and 2parts of sodium hydroxide in 4 parts of water was added dropwise. Thesolid was filtered and crystalized with methanol and a mixtue of ethylacetate and ether to give 2.6 parts of white solid. The structure of theproduct was confirmed on the basis of ¹H NMR. The reaction proceeded asfollows:

-   (e) Photooxidation of [(3-Phosphoryloxyphenyl) (2-phenoxyethoxy)    methylene]-5-chloroadamantane, disodium salt (66).

Alkene (66) was photooxidized as reported above, to give[4-(2-phenoxyethoxyl)-4-(3-phosphoryloxyphenyl)] spiro[1,2-dioxetane-3,2′-5-chloroadamantane], disodium salt (67).

EXAMPLE VIII

This example illustrates the preparation of[(4-Methoxy)-4-(3-phosphoryloxy-4-chlorophenyl)] spiro[1,2-dioxetane-3,13′-tricyclo [7.3.1,o^(2,7)] tridec-2,7-ene], disodiumsalt (74). The sequence of the reactions in accordance herewith:

-   (a) Synthesis of methyl 4-chloro-3-hydroxybenzoate (68)

In a 250 mL round bottom flask 5 parts of 4-chloro-3-hydroxybenzoic acidwas dissolved in 100 mL of methanol and 25 drops of concentratedsulfuric acid was added. The reaction mixture was heated under refluxedfor 24 hours. TLC on silica gel shows the formation of new product. Thesolvent was evaporated and solid was dissolved in 150 mL of ethylacetate. The organic layer was washed with 100 ml of 5% solution ofsodium bicarbonate and dried over sodium sulfate. The solvent wasevaporated under reduced pressure to give a white solid, yield 5.0 g.

-   (b) Synthesis of methyl 3-tert-butyldimethylsiloxy-4-chlorobenzoate    (70).

Into a 250 mL round bottom flask was added 40 mL of dry dimethylformamide. Five parts of benzoate (69) and 4.86 parts oftert-butyldimethyl silyl chloride were added to the reaction flask withstirring. 4.04 parts of Imidazole was added in portions and stirring wascontinued for 12 hours. The reaction mixture was diluted with 50 mLwater and extracted with 200 mL of ethyl acetate. The organic layer waswashed twice with 100 mL of water and dried on anhydrous sodium sulfate.The solvent was evaporated under reduced pressure to give an oil, yield7.1 parts. This material was pure enough for the next step of thereaction. The material was prepared according to the following:

-   (c) Synthesis of (3-tert-butyldimethylsiloxy-4-chlorophenyl)    methoxymethylene tricyclo[7.3.1.0^(2,7)] tridec-2,7-ene (71).

Into a 500 mL three-neck flask equipped with magnetic stirrer,pressure-equalizing addition funnel and nitrogen line was charged with150 mL of anhydrous THF. Ttitanium tetrachloride (12 mL) was addeddropwise over a period of 30 minutes. The suspension was stirred for 20minutes and 18 parts of zinc was added in small portions. The reactionmixture was heated under reflux for 2 hours and 29 mL of triethylaminewas added dropwise. After refluxing one hour, 6 parts of a solution ofmethyl 3-tert-butyldimethylsiloxy-4-chlorobenzoate and 4 parts of ketone(35) in 35 mL of dry THF was added over a period of 60 minutes and thereaction mixture was heated for one hour. The mixture on TLC silia gelplate showed the presence of the starting ester. An additional 2.5 partsof starting ketone (35) in 35 mL of dry THF was added dropwise over 45minutes and refluxed for two hours. The mixture was cooled to roomtemperature, diluted with 500 mL of hexane and decanted. The residue waswashed with 3×200 mL of hexane. The combined hexane layer was filteredand evaporated under reduced pressure to give an oily material which waspurified by chromatography on silica gel column using 5% ethylacetate/hexane as an eluant. The fractions were checked by TLC on silicagel plate and the desired fractions were combined. The solvent wasevaporated under reduced pressure to give an oil, yield 5.6 parts. Thestructure was confirmed by ¹H NMR. The reaction proceeded as follows:

-   (d) Synthesis of (3-hydroxy-4-chlorophenyl) methoxymethylene    tricyclo[7.3.1.0^(2,7)] tridec-2,7-ene (72).

To a crude solution of 5.6 parts of(3-tert-Butyldimethylsiloxy-4-chlorophenyl) methoxymethylenetricyclo[7.3.1.0^(2,7)] tridec-2,7-ene in 100 mL of THF was added 7parts of 70% tetra-n-butylammonium fluoride in 30 mL of THF over aperiod of 10 minutes and stirring was continued for two hours. TLC onsilica gel plate showed the formation of new product. Solvent wasevaporated under reduced pressure and the oily material was dissolved in200 mL of methylene chloride and washed with 2×100 mL of water. Afterdrying over anhydrous sodium sulfate, the solvent was evaporated and theoily product was purified on silica gel column. TLC on silica gel plateof the fractions were checked and the combined organic solvent wasevaporated, yield 3.25 parts. The structure of the product was confirmedon the basis of ¹H NMR. The reaction proceeded as follows:

-   (e) Synthesis of (3-phosphoryloxy-4-chlorophenyl) methoxymethylene    tricyclo [7.3.1.0^(2,7)] tridec-2,7-ene, disodium salt (73).

Into a 100 mL three-neck round bottom flask equipped with mageneticstirrer was added 15 mL of dry methylene chloride under nitrogen andthen 1.57 mL of phosphorous oxychloride was added dropwise (reactionflask was cooled in ice-water bath). A solution of 3.0 parts of alkene(72) in 15 mL of methylene chloride containing 1.57 parts of anhydrouspyridine was added to the reaction flask over a period of 30 minutes.The reaction mixture was stirred at room temperature for 3 hours. TLC onsilica gel plate showed the formation of new product. A solutioncontaining 3.56 parts of 3-hydroxypropionitrile and 3.56 parts ofanhydrous pyridine in 35 ml of methylene chloride was added dropwise tothe reaction mixture over 25 minutes and was stirred for 15 hours atroom temperature. The reaction was cooled to ice-water temperature andthe solid was filtered, washed with cold methylene chloride. The solventwas evaporated and oily material was chromatographed on silica gelcolumn using 70% ethyl acetate/hexane containing 0.1% triethyl amine.Fractions were checked by TLC on silica gel plate and the desired:fractions were combined and evaporated under reduced pressure to give anoil. The oily material was dissolved in 150 mL of acetone and a solutionof 2.5 parts of sodium hydroxide in 5 mL of water was added dropwise.Stirring was continued for two hours and the solid was filtered andwashed with acetone. The soild material was crystalized with methanoland acetone mixture. The solid was filtered and washed with acetone anddried, yield 3.15 parts. The structure was confirmed on the basis of ¹HNMR. The reaction proceeded as follows:

-   (f) Photooxidation of (3-phosphoryloxy-4-chlorophenyl)    methoxymethylene tricyclo [7.3.1.02,7] tridec-2,7-ene, disodium salt    (73).

Alkene (73) was photooxidized as reported above to give[(4-Methoxy)-4-(3-phosphoryloxy-4-chlorophenyl)] spiro[1,2-dioxetane-3,13′-tricyclo [7.3.1,o^(2,7)] tridec-2,7-ene], disodiumsalt (74).

EXAMPLE IX

This example illustrates the preparation of[(4-Methoxy)-4-(3-phosphoryloxyphenyl)] spiro[1,2-dioxetane-3,9-(2,4-diphenylbicyclo[3.3.1]non-2-ene)], disodium salt(81). The sequence of the reactions in accordance herewith:

-   (a). Synthesis of 2-(α-phenyl-β-benzoylethyl)-cyclohexanone (76).

Ten parts of benzalacetophenone and 5 parts of cyclohexanone wasdissolved in 200 mL of ethyl alcohol and a solution of 5 parts of sodiumhydroxide in 100 ml of ethanol was added to the reaction mixture. After30 minutes the solution became cloudy and white solid was appeared. Thereaction mixture was stirred for 10 hours at room temperature and solidwas filtered, washed with water and then with 100 ml of ethyl alcohol.The white solid was dried under reduced pressure, with a yield of 12parts. The reaction proceeded as follows:

-   (b). Synthesis of 2,4-Diphenyl bicyclo[3.3.1]non-2-ene-9one (77).

Into a 1 L round bottom flask fifteen parts of2-(α-phenyl-β-benzoylethyl)-cyclohexanone, 500 ml acetic acid and 100 mLof concentrated hydrochloric acid was added. Reaction mixture was heatedunder reflux for 45 hours. The hot reaction mixture was diluted with 250ml of water (reaction mixture was cloudy). The reaction mixture was coolto room temperature to give a white solid which was filtered and driedunder vacuum, to yield 11.75 g of product. The reaction proceeded asfollows:

-   (c) Synthesis of    (3-tert-Butyldimethylsiloxyphenyl)methoxymethylene-2,4-diphenyl    bicyclo[3.3.1]non-2-ene (78).

Into a 1000 mL three-neck flask equipped with magnetic stirrer,pressure-equalizing addition funnel and nitrogen line was charged with200 mL of anhydrous THF. Ttitanium tetrachloride (17 mL) was addeddropwise over a period of 30 minutes. The suspension was stirred for 20minutes and 25.5 parts of zinc was added in small portions. The reactionmixture was heated under reflux for 2 hours and 42.5 mL of triethylaminewas added dropwise. After refluxing one hour, 9 parts of a solution ofmethyl 3-tert-butyldimethylsiloxybenzoate and 4 parts of ketone (77) in100 mL of dry THF was added over a period of 60 minutes and the reactionmixture was heated for one hour. The mixture on TLC silica gel plateshowed the presence of the starting ester. An additional 1 part ofstarting ketone (77) in 35 mL of dry THF was added dropwise over 45minutes and refluxed for two hours. The mixture was cooled to roomtemperature, diluted with 600 mL of hexane and decanted. The residue waswashed with 3×250 mL of hexane. The combined hexane layer was filteredand evaporated under reduced pressure to give an oily material which waspurified by chromatography on silica gel column using 5% ethylacetate/hexane as an eluant. The fractions were checked by TLC on silicagel plate and the desired fractions were combined. The solvent wasevaporated under reduced pressure to give an oil, yield 9.6 parts. Thestructure was confirmed by ¹H NMR. The reaction proceeded as follows:

-   (c) Synthesis of (3-Hydroxyphenyl)methoxymethylene-2,4-diphenyl    bicyclo[3.3.1]non-2-ene (79).

To a solution of 9.6 parts of (3-tert-Butyldimethylsiloxyphenyl) methoxymethylene-2,4-diphenyl bicyclo[3.3.1]non-2-ene (78) in 100 mL of THF wasadded 9 parts of 70% tetra-n-butylammonium fluoride in 30 mL of THF overa period of 10 minutes and stirring was continued for two hours. TLC onsilica gel plate showed the formation of new product. Solvent wasevaporated under reduced pressure and the oily material was dissolved in300 mL of methylene chloride and washed with 2×100 mL of water. Afterdrying over anhydrous sodium sulfate, the solvent was evaporated and theoily product was purified on silica gel column. TLC on silica gel plateof the fractions were checked and the combined organic solvent wasevaporated, yield 6.24 parts. The structure of the product was confirmedon the basis of ¹H NMR. The reaction proceeded as follows:

-   (d) Synthesis of (3-Phosphoryloxyphenyl)    methoxymethylene-2,4-diphenyl bicyclo[3.3.1]non-2-ene, disodium salt    (80).

Into a 100 mL three-neck round bottom flask equipped with mageneticstirrer was added 40 mL of dry methylene chloride under nitrogen andthen 3.76 mL of phosphorous oxychloride was added drop-wise (reactionflask was cooled in ice-water bath). A solution of 5 parts of alkene(79) in 30 mL of methylene chloride containing 2.14 parts of anhydrouspyridine was added to the reaction flask over a period of 30 minutes.The reaction mixture was stirred at room temperature for 3 hours. TLC onsilica gel plate showed the formation of new product. A solutioncontaining 4.86 parts of 3-hydroxypropionitrile and 4.86 parts ofanhydrous pyridine in 30 ml of methylene chloride was added dropwise tothe reaction mixture over 25 minutes and was stirred for 48 hours atroom temperature. The reaction was cooled to ice-water temperature andthe solid was filtered, washed with cold methylene chloride. The solventwas evaporated and oily material was chromatographed on silica gelcolumn using 70% ethyl acetate/hexane containing 0.1% triethyl amineFractions were checked by TLC on silica gel plate and the desiredfractions were combined and evaporated under reduced pressure to give anoil. The oily material was dissolved in 300 mL of acetone and a solutionof 7 parts of sodium hydroxide in 15 mL of water was added dropwise.Stirring was continued for two hours and the solid was filtered andwashed with acetone. The soild material was crystalized with methanoland acetone mixture. The solid was filtered and washed with acetone anddried, yield 4.85 parts. The structure was confirmed on the basis of ¹HNMR. The reaction proceeded as follows:

-   (e) Photooxidation of (3-Phosphoryloxyphenyl)    methoxymethylene-2,4-diphenyl bicyclo[3.3.1]non-2-ene, disodium salt    (80).

Alkene (80) was photooxidized as reported above, to give [(4Methoxy)-4-(3-phosphoryloxyphenyl)] spiro[1,2-dioxetane-3,9-(2,4-diphenylbicyclo[3.3.1]non-2-ene)], disodium salt(81).

1. An alkene intermediate corresponding to the formula:

wherein R₇ is selected from the group consisting of alkyl, aryl,aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl,alkyletheralkyl, alkyletheraryl, alkyl (etheralkyl)₂, alkyl(etheralkyl)₃, alkyletherhaloalkyl, alkyl(etherhaloalkyl)₂, alkylalkene,alkylalkyne, arylalkene, arylalkyne, halogenated alkyl (mono, di, tri orany position in normal or branched or cyclic chain), alkylalcohol,alkylnitrile, alkylamine, alkylacid, (mono or dibasic) or the inorganicsalts thereof, haloalkylalcohol, haloalkylnitrile, haloalkylamine,haloalkylacid, mono or dibasic, or the inorganic salts thereof, alinker-fluorescent molecule, a linker-antibody , a linker-antigen, alinker-biotin, a linker-avidin, a linker-protein, a linker-carbohydrateor a linker-lipid; Ar is aryl and may be phenyl, substituted phenyl,naphthyl, substituted naphthyl, anthryl, substituted anthryl, or otherfluorescent or non-fluorescent aromatic group; Y is a hydrogen, alkyl,acetate, t-butyldimethylsilyl or an enzyme cleavable group, or anantibody cleavable group and R is hydrogen, alkyl or substituted alkyl,aryl or substituted aryl, alkylaryl or substituted alkylaryl, arylalkylor substituted arylalkyl; X is chloro or hydrogen.
 2. The alkene ofclaim 1 which is any of: a. [(3-Phosphoryloxyphenyl)methoxymethylene-8-n -propyltricyclo[7.3.1.0^(2,7)) tridec-2,7-ene],disodium salt; b. [(3-Phosphoryloxyphenyl) methoxymethylenetricyclo[7.3.1.0^(2,7)) tridec-2,7-ene], disodium salt; c.[(3-Phosphoryloxyphenyl) (2,2,2-trifluoroethyoxy)methylene]tricyclo[7.3.1.0^(2,7)) tridec-2,7-ene], disodium salt; d.[(3-Phosphoryloxyphenyl) (2-phenoxyethoxy)methylene]tricyclo[7.3.1.0^(2,7)) tridec-2,7-ene], disodium salt; e.[(3-Phosphoryloxy-4-chlorophenyl) methoxymethylenetricyclo[7.3.1.0^(2,7)) tridec-2,7-ene], disodium salt, and mixturesthereof.
 3. An alkene intermediate corresponding to the formula:

where R₇ is selected from the groups consisting of alkyl, aryl, aralkyl,alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl,alkyletheralkyl, alkyletheraryl, alkyl(etheralkyl)₂, alkyl(etheralkyl)₃,alkyletherhaloalkyl, alkyl(etherhaloalkyi)₂, alkylalkene, alkylalkyne,arylalkene, arylalkyne, halogenated alkyl(mono, di, tri or any positionin normal or branched or cyclic chain), alkylalcohol, alkylnitrile,alkylamine, alkylacid (mono or dibasic) or the inorganic salts thereof,haloalkylalcohol, haloalkylnitrile, haloalkylamine, haloalkylacid (monoor dibasic) or inorganic salts thereof, a linker-fluorescent molecule, alinker-antibody, a linker-antigen, a linker-biotin, a linker-avidin, alinker-protein, a linker-carbohydrate or a linker-lipid; where Ar isaryl and may be phenyl, substituted phenyl, naphthyl, substitutednaphthyl, anthryl, substituted anthryl, or other fluorescent ornon-fluorescent aromatic group; Y is a hydrogen, alkyl, acetate,t-butyldimethylsilyl or an enzyme cleavable group or an antibodycleavable group; X is chloro or hydrogen in the benzene ring; Z ishydrogen and R is hydrogen, alkyl or substituted alkyl, aryl orsubstituted aryl, alkylaryl or substituted alkylaryl, arylalkyl orsubstituted arylalkyl is substituted in the bicyclo[3.3.1] nonane ring.4. The alkene intermediate of claim 3 which is: [(3-Phosphoryloxyphenyl)methoxymethylene-2,4-diphenyl bicyclo[3.3.1]non-2-ene, disodium salt. 5.An alkene intermediate, for the formation of a stable 1,2-dioxetane,corresponding to the following formula:

(1) when Ar—O—Y and OR₇ join together to give an aryl group substitutedwith an Y-oxy group to form an alkene intermediate for the formation ofa stable 1,2-dioxetane which is triggerable to form an unstableintermediate oxide, R₅ and R₆ form either (a) an adamantene ring with orwithout side chain, the side chain being with or without at least oneheteroatom, wherein the carbon atom of the alkene intermediate that islinked to R₅ and R₆ is a ring carbon atom of the adamantene ring or (b)Ar₁—R_(x), where R_(x) is an adamantene ring containing a substituted oran unsubstituted fused aromatic ring or a substituted or anunsubstituted aromatic ring attached by a linker arm, wherein the carbonatom of the alkene intermediate that is linked to R₅ and R₆ is a ringcarbon atom of the adamantene ring; or (2) when Ar—O—Y and OR₇ do notjoin together, (a) Ar is aryl and may be phenyl, substituted phenyl,naphthyl, substituted naphthyl, anthryl, substituted anthryl or otherfluorescent or non-fluorescent aromatic group; Y is either hydrogen,alkyl, acetate, t-butyldimethylsilyl or an enzyme cleavable group or anantibody cleavable group; R₇ is selected from the group consisting ofalkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl,cycloheteroalkyl, alkyletheralkyl, alkyletheraryl, alkyl(etheralkyl)₂,alkyl(etheralkyl)₃, alkyletherhaloalkyl, alkyl(etherhaloalkyl)₂,alkylalkene, alkylalkyne, arylalkene, arylalkyne, halogenatedalkyl(mono, di, tri or any position in normal or branched or cyclicchain), alkylalcohol, alkylnitrile, alkylamine, alkylacid (mono ordibasic) or the inorganic salts thereof, haloalkylalcohol,haloalkylnitrile, haloalkylamine, haloalkylacid (mono or dibasic) orinorganic salts thereof, a linker-fluorescent molecule, alinker-antibody, a linker-antigen, a linker-biotin, a linker-avidin, alinker-protein a linker-carbohydrate or a linker-lipid, and R₅ and R₆form either (i)

which is an adamantene ring with or without a side chain, the side chainbeing with or without at least one heteroatom, wherein the carbon atomof the alkene intermediate that is linked to R₅ and R₆ is a ring carbonatom of the adamantene ring or (ii) Ar₁—R_(x) which is an adamantenering containing a substituted or an unsubstituted fused aromatic ring ora substituted or an unsubstituted aromatic ring attached by a linkerarm, wherein the carbon atom of the alkene intermediate that is linkedto R₅ and R₆ is a ring carbon atom of the adamantene ring or, (b) Ar isaryl and may be phenyl, substituted phenyl, naphthyl, substitutednaphthyl, anthryl, substituted anthryl, or other fluorescent ornon-fluorescent aromatic group; Y is a hydrogen, alkyl, acetate,t-butyldimethylsilyl, an enzyme cleavable group or an antibody cleavablegroup; R₇ is selected from the group consisting of alkyletheralkyl,alkyletheraryl, alkyl(etheralkyl)₂, alkyl (etheralkyl)₃,alkyletherhaloalkyl, alkyl (etherhaloalkyl)₂, alkylalkene, alkylalkyne,arylalkene, arylalkyne, halogenated alkyl(mono, di, tri or any positionin normal or branched or cyclic chain), alkylalcohol, alkylnitrile,alkylamine, alkylacid (mono or dibasic) or the inorganic salts thereof,haloalkylalcohol, haloalkylnitrile, haloalkylamine, haloalkylacid (monoor dibasic) or the inorganic salts thereof, a linker-fluorescentmolecule, a linker-antibody, a linker-antigen, a linker-biotin, alinker-avidin, a linker-protein or a linker-carbohydrate orlinker-lipid; and R₅ and R₆ form

which is an adamantine, wherein the carbon atom of the alkeneintermediate that is linked to R₅ and R₆ is a ring carbon atom of theadamantene ring, or (c) Ar is aryl and may be phenyl, substitutedphenyl, naphthyl, substituted naphthyl, anthryl, substituted anthryl orother fluorescent or non-fluorescent aromatic group; Y is a hydrogen,alkyl, acetate, t-butyldimethylsilyl or enzyme cleavable group, or anantibody cleavable group; R₇ is selected from the group consisting ofalkyletheralkyl, alkyletheraryl, alkyl(etheralkyl)₂, alkyl(etheralkyl)₃,alkyletherhaloalkyl, alkyl(etherhaloalkyl)₂, alkylalkene, alkylalkyne,arylalkene, arylalkyne, halogenatedalkyl(mono, di, tri or position innormal or branched or cyclic chain), alkylalcohol, alkylnitrile,alkylamine, alkylacid (mono or dibasic) or the inorganic salts thereof,haloalkylalcohol, haloalkylnitrile, haloalkylamine, haloalkylacid (monoor dibasic) or inorganic salts, a linker-fluorescent molecule, alinker-antibody, a linker-antigen, a linker-biotin, a linker-avidin, alinker-protein, a linker-carbohydrate or a linker-lipid; R₅ and R₆ forman adamantene which can contain halogens and hetero atoms in the ring orside chain thereof, and wherein the carbon atom of the alkeneintermediate that is linked to R₅ and R₆ is a ring carbon atom of theadamantene ring.
 6. The alkene intermediate of claim 5 corresponding tothe formula:

wherein R₇ has at least one hetero atom with either a saturated or anunsaturated organic group with or without substitution, and is selectedfrom the group consisting of alkyletheralkyl, alkyletheraryl, alkyl(etheralkyl)₂, alkyl(etheralkyl)₃, alkyletherhaloalkyl,alkyl(etherhaloalkyl)₂, alkylalkene, alkylalkyne, arylalkene,arylalkyne, halogenated alkyl (mono, di, tri or any position in normalor branched or cyclic chain), alkylalcohol, alkylnitrile, alklamine,alkylacid (mono or dibasic) or the inorganic salts thereof,haloalkylalcohol, haloalkylnitrile, haloalkylamine, haloalkylacid (monoor dibasic) or inorganic salts thereof, a linker-fluorescent molecule, alinker-antibody a linker-antigen, a linker-biotin, a linker-avidin, alinker-protein, a linker-carbohydrate or a linker-lipid; Ar is aryl andmay be phenyl, substituted phenyl, naphthyl, substituted naphthyl,anthryl, substituted anthryl, or other fluorescent or non-fluorescentaromatic group, Y is a hydrogen, alkyl, acetate, tert-butyldimethylsilylor an enzyme cleavable group or an antibody cleavable group, R ishydrogen, alkyl or substituted alkyl, aryl or substituted aryl,alkylaryl or substituted alkylaryl, arylalkyl or substituted arylalkylin phenyl ring, X is choro-or hydrogen and Z is hydrogen.
 7. The alkeneintermediate of claim 5 corresponding to the formula:

where R₇ is selected from the groups consisting of alkyl, aryl, aralkyl,alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl,alkyletheralkyl, alkyletheraryl, a alkyl (etheralkyl)₂,alkyl(etheralkyl), alkyletherhaloalkyl, alkyl(etherhaloalkyl)₂,alkylalkene, alkylalkyne, arylalkene, arylalkyne, halogenatedalkyl(mono, di, tri or any position in normal or branched or cyclicchain), alkylalcohol, alkylnitrile, alkylamine, alkylacid (mono ordibasic) or the inorganic salts thereof, haloalkylalcohol,haloalkylnitrile, haloalkylamine, haloalkylacid (mono or dibasic) orinorganic salts thereof, a linker-fluorescent molecule, alinker-antibody, a linker-antigen, a linker-biotin, a linker-avidin, alinker-protein, a linker-carbohydrate or a linker-lipid; Ar is aryl andmay be phenyl, substituted phenyl, naphthyl, substituted naphthyl,anthryl, substituted anthryl, or other fluorescent or non-fluorescentaromatic ; Y is a hydrogen, alkyl, acetate, t-butyldimethyl silyl, anenzyme cleavable group or an antibody cleavable group, X is chloro orhydrogen and Z is hydrogen.
 8. The alkene intermediate of claim 5wherein R₅ and R₆ each contain a substituent selected from the groupconsisting of halogen or hetero atoms in the ring or side chain thereof.9. The alkene intermediate of claim 5 which is: (3-Phosphoryloxyphenyl)methoxymethylene adamantan-4,5-ene, disodiumn salt.